The effectiveness of nucleoside analogs used to treat HIV infections is limited by the evolution of resistance by HIV reverse transcriptase (RT), and by toxicity due to incorporation into mitochondrial DNA by the human mitochondrial DNA polymerase. The clinically observable effectiveness versus toxicity of nucleoside analogs can be understood at the molecular level in terms of the discrimination against these analogs during DNA polymerization catalyzed by HIV RT relative to that by the human mitochondrial DNA polymerase. Understanding polymerase specificity is at the heart of the problems inherent in developing less toxic and more effective nucleoside analogs. This application is a continuation of our studies to understand nucleotide selectivity by the human mitochondrial DNA polymerase and its role in the toxicity of nucleoside analogs. In collaboration with Whitney Yin, we will solve the structure of the mitochondrial DNA polymerase and use the new structural information to examine the efficiency and specificity of nucleotide incorporation. We will use site-directed mutagenesis and comprehensive kinetic analysis to evaluate the roles of individual amino acids. We will examine mutants in the mitochondrial polymerase gene that are linked to heritable diseases and attempt to correlate changes in structure and function of the polymerase to the physiological effects of mutations. These studies will provide additional data to understand the physiological basis for the toxicity of nucleoside analogs, and the role of mutations and oxidative damage in ageing. Initiation of DNA polymerization at the mitochondrial replication origin will be studied using synthetic RNA/DNA duplex. We will also reconstitute the replisome using the mitochondrial helicase with the polymerase to examine leading strand synthesis. The roles of the accessory protein and the possible involvement of p53, a tumor suppressor, in regulating polymerase activity will be assessed. We will use pre-steady state and single turnover rapid kinetic studies to directly examine reactions occurring at the active. These methods enable reaction pathways to be established and reaction rates to be quantified by direct measurement and these kinetic parameters can be related directly to structure. This research will provide a better understanding of the role of the mitochondrial polymerase in diseases related to mitochondrial function, and will provide new information to define the molecular basis for nucleotide discrimination by the human mitochondrial DNA polymerase, and it will facilitate the continued development of more effective, less toxic nucleoside analog to combat HIV infections. PUBLIC HEALTH RELEVANCE The effectiveness of nucleoside analogs used to treat HIV infections is limited by their toxicity due to incorporation into mitochondrial DNA by the human mitochondrial DNA polymerase. This research will provide a better understanding of the role of the mitochondrial polymerase in diseases related to mitochondrial function, will provide new information to define the molecular basis for nucleotide discrimination, and will facilitate the development of more effective, less toxic nucleoside analog to combat HIV infections.